Because of its broad applicability, oligonucleotide-directed site-specific mutagenesis embodies a tool fundamental to modern molecular biology research. Its utilization for the introduction of insertions, deletions, and substitutions into coding or non-coding DNA permits the execution of a wide variety of investigations including analysis of structure-function relationships at the level of DNA, RNA, and protein. In the area of enzyme catalysis for example, structural and mechanistic information derived from amino acid substitutions by site-directed mutagenesis continues to add significantly to a wealth of knowledge obtained from biochemical and biophysical studies.
Following the initial reports demonstrating the use of synthetic oligonucleotides to create phenotypically selectable site-specific mutations within .phi.X174 DNA, general methods for site-directed mutagenesis were put forth. Since these developments around ten years ago, many different approaches designed to reduce the time and effort necessary to construct a desired site-specific mutation have been described. In general, these methods fall into two categories: those designed to eliminate the laborious differential hybridization manipulations necessary for identification of the mutant molecules against a large background of parental forms, and the others, aimed at circumventing the requirement for cloning of the target DNA into specialized vectors for the production of single-stranded DNA templates.
An approach which maintains general applicability and high efficiency, and allows site-directed mutagenesis to be performed directly on any existing plasmid would therefore be desirable.